This project seeks to understand the functional role of latent transforming growth factor beta binding protein (LTBP4) in the pathogenesis of muscle disease, specifically the muscular dystrophies. Muscular dystrophy arises from ongoing muscle degeneration and insufficient regeneration, leading to loss of muscle with replacement by scar or fibrosis resulting in muscle weakness. LTBP4 is a member of a family of proteins that regulate the bioavailability of transforming growth factor (TGF)2 proteins. Work by others has shown that enhanced TGF2 signaling in skeletal muscle contributes to impaired regeneration in muscle, however the molecular mechanism underlying enhanced TGF2 activity is understood. We identified a DNA variant in the proline-rich region of murine LTBP4 that segregates with mild and severe forms of muscular dystrophy in mice. Our data support that this region of LTBP4 is modified by proteolysis and that this proteolysis regulates TGF2 release from the extracellular matrix. In this model, release of TGF2 from the extracellular matrix leads to increased activation of TGF2 signaling, enhanced muscle degeneration, and reduced muscle regeneration. Furthermore, we identified non-synonymous single nucleotide polymorphisms (SNPs) in crucial protein domains of human LTBP4. We predict that these proteins variants will alter TGF2 availability, thus providing a mechanism to explain variable cardiac and muscle function among patients. We have begun screening the human LTBP4 gene from unrelated human DNA panels for normal variations in the nucleotide sequence. LTBP4 variants will be a valuable tool in predicting disease prognosis. Given the larger deletion of the human LTBP4 proline-rich region compared to murine LTBP4, we hypothesize that the human LTBP4 will be more susceptible to proteolytic cleavage, resulting in enhanced TGF2 activity. We plan to test this by expressing fragments of LTBP4 in vitro and determining proteolytic susceptibility. We will also test whether the SNP variants affect proteolytic susceptibility and TGF2 signaling. In order to determine the role of human LTBP4 in modifying muscular dystrophy outcome, we will express human LTBP4 and determine how this affects mouse models of muscular dystrophy. Elucidating the role of LTBP4 in TGF2 bioavailability and signal transduction is paramount to understanding muscular dystrophy pathogenesis. PUBLIC HEALTH RELEVANCE: Increased TGF2 activity has been shown to aggravate muscle disease, however the molecular mechanism underlying enhanced TGF2 activity remains poorly understood. In this project we propose a novel mechanism by which mutations in the latent transforming growth factor binding protein 4 (LTBP4) alter TGF2 bioavailability and activity. Our studies will help extend our understanding of the genetic bases of muscle disease and assist the development of treatment options.